A method of detecting and quantifying immunoglobulin E-antibodies in serum is disclosed in the brochure "Specific IgE, Magic.RTM. Lite SQ.TM., published by Ciba Corning Diagnostics Corp. and ALK Laboratories in September 1990. In said method a specific allergen covalently bound to paramagnetic particles reacts with the allergen-specific IgE-antibody in a serum or plasma sample. After a first incubation period and washing away unbound non-specific IgE, a chemiluminescent acridinium ester-labelled monoclonal antibody against IgE is added. Following a second incubation period the solid phase bound and labelled antibody is measured in a Magic Lite.RTM. Analyzer (Cat. No. 472733 or Cat. No. 472270) which automatically injects reagents, which initiate the chemiluminescent reaction. When using said method only the final step of initiating and measuring the chemiluminescence can be automated. The chemiluminescent acridinium ester labelling compound is described in U.S. Pat. No. 4,745,181.
U.S. Pat. No. 4,946,958 discloses a chemiluminescent acridinium ester linked to an N-succinimidyl moiety which can be further linked to a protein or polypeptide to provide an immunologically reactive luminescent reagent. Said reagent can be used in an immuno-assay, which may involve the simultaneous binding of a solid phase antibody, an antigen molecule and a labelled anti-antibody, separation and washing of the solid phase and quantifying the luminescence of the solid phase.
Strasburger et al. discloses in Methods in Enzymology, 184(1990), pp 481-496 the use of a two-site chemiluminescent immuno-assay, wherein hGH and hCG hormones are captured by antibodies immobilized on microtiter plates and labelled with a chemiluminescent agent coupled to avidin through a biotin labelled second antibody. Antigenic analytes, such as protein hormones, may be assayed directly from serum samples and compared to standard curves. The concentration of immunoglobulins, such as IgE, is, however, extremely patient dependent and an assay of a specific IgE must be compared with the individual patient level of total IgE and therefore requires an assay having a greater dynamic range than is obtainable in the assay disclosed by Strasburger et al.
EP-A-0 425 217 discloses a hybridization assay wherein a chemiluminescent complex is formed comprising a nucleic acid hybridized with a first labelled nucleotide probe coupled to paramagnetic particles and a second nucleotide probe labelled with biotin and coupled to an avidin-acridinium ester. However, the person skilled in the art confronted with the problem of providing a fully automated method of detecting antibodies will search for a method which can be carried out in one reaction container and preferably under ambient reaction conditions. The assay represented here is fundamentally different as it employs detection of specific nucleotide sequences, which are not antigens towards which specific antibodies can be raised. Moreover, it is necessary in said assay to use elevated temperatures for the hybridization reaction and a hapten-oligonucleotide probe which is not necessary nor desirable in the immuno-assays.
Until now immuno-assays for the quantification of immunologically active molecules, such as immunoglobulins (e.g. specific immunoglobulin-E), in biological fluids, such as serum, have been manual, e.g. the Enzyme Linked Immuno Sorbent Assay (ELISA), or semi-automatical. And the typical duration of a semi-automatical immunoassay, such as the Magic.RTM. Lite SQ.TM. specific IgE assay referred to above, is approximately two hours.
Moreover, commercial specific IgE assays (CAP,RAST, supplied by Pharmacia, Uppsala) and the Magic.RTM. Lite SQ.TM. specific IgE assay use a total-IgE (i.e. the total amount of antibodies present in the immunoglobulin class of IgE including all immunoglobulin subclasses) reference assay having a non-identical protocol resulting in unprecise data. Only assays using the same catching and detection procedures are directly comparable. For example all specific IgE close response curves must be parallel with total IgE response curves. The reason is that the required dynamic range for a specific (or total?) IgE assay is 2 decades and the required dynamic range for a total IgE assay is from 2 to 7 decades, and the existing immuno-assays do not allow concentration measurements over the entire range. Thus, until now it has been necessary to use different protocols for the specific and total immunoglobulin assays having different reagents.
Because of the increasing interest in safe laboratory procedures there is a need for a fully automated method of detecting substances, such as antibodies, in biological fluids, such as human serum, plasma, blood, milk, urine or saliva, which method should provide a minimized risk of contact with hazardous fluids. Also, the increasing use of laboratory tests in diagnostics calls for methods of short duration, preferably only a few minutes.
It is therefore an object of this invention to provide a method of detecting an antibody in a sample, which method is safe, rapid, and fully automatable.